EP2880154A1 - Production of infectious influenza viruses - Google Patents
Production of infectious influenza virusesInfo
- Publication number
- EP2880154A1 EP2880154A1 EP13742451.1A EP13742451A EP2880154A1 EP 2880154 A1 EP2880154 A1 EP 2880154A1 EP 13742451 A EP13742451 A EP 13742451A EP 2880154 A1 EP2880154 A1 EP 2880154A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- influenza
- virus
- cells
- viruses
- promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/00051—Methods of production or purification of viral material
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- C12N2760/00011—Details
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- C12N2760/00052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
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- C12N2760/00011—Details
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- C12N2760/16161—Methods of inactivation or attenuation
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16251—Methods of production or purification of viral material
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
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- C12N2760/16261—Methods of inactivation or attenuation
- C12N2760/16264—Methods of inactivation or attenuation by serial passage
Definitions
- Type C viruses have only one multifunctional surface glycoprotein, "hemagglutinin- esterase-fusion protein” (HEF).
- HEF hemagglutinin- esterase-fusion protein
- RNA POL I promoter is generally used in the plasmids that allow the production of influenza vRNAs
- human and simian cells are the most appropriate cell lines to be used as transfected cell line in the reverse genetics system.
- Homologous recombination involves a process of breakage and reunion in regions of identical DNA sequences between two DNA molecules to result in new combinations of genetic materiel (Watt et al, 1985, Proc Natl Acad Sci, 82:4768-4772).
- These previously described recombinational cloning systems are based on a 25 nucleotides recombination cassette comprising the consensus 5' (Uni13) and 3' (Uni12) conserved non-coding ends of influenza A segments between human POL I promoter and terminator. They allow the rapid and direct cloning of any influenza A genome.
- the nucleotide sequences of vRNA 5' and 3' non-coding ends of influenza B genomes are different from influenza A virus, influenza B genomes cannot be cloned based on this recombination cassette.
- the term "reassortant virus” denotes a virus which contains genetic material that results from the combination of genetic material of at least two donor viruses.
- its genetic material usually contains at least the HA and NA genes from a seasonal or pandemic virus whereas the other genes (backbone genes) are from one or several other donor viruses which have been selected for their ability to grow easily on the substrate of production used for manufacturing the flu vaccine (such as the allantoic cavity of embryonated hen's eggs or a permissive cell line) and/or to be less or non pathogen for the humans.
- donor viruses that contribute as "provider" of backbone genes include A/Puerto Rico/8/34 (H1 N1 ) (A/PR/8/34), B/Lee/40 and/or B/Panama/45/90 viruses.
- said promoter which binds to rodent RNA polymerase I comprises, or consists of the sequence SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
- said negative single-stranded RNA virus may be a reassortant virus and/or chimeric virus. These viruses can be attenuated virus, or inactivated virus.
- the cloning strategy involves homologous recombination between the vector according to the invention and the cDNA sequence to be cloned.
- the obtained cDNA comprises, in the 5' to 3' sense, a nucleotide sequence from the promoter that binds to human polymerase I, the cDNA encoding a vRNA of a virus and a nucleotide sequence from the hepatitis delta ribozyme sequence. Consequently, at step (iii) the obtained cDNA at step (i) is cloned in antisens into the vector according to the invention.
- the seed of infectious influenza viruses is obtained by transfecting cells with a set of expression vectors capable of generating said infectious viruses.
- the cell line used for the purpose of the invention is a CHO cell line which does not express on its surface the Sia2- 6Gal receptors, but express weakly the Sia 2-3Gal receptors such that less than 50% of the cell population is fluorescent in presence of the digoxigenin-labeled Maackia amurensis agglutinin.
- the cell line used for the purpose of the invention is the CHO-K1 cell line, in particular the cell line referenced at the ATCC under the number CCL-61 .
- the CHO-K1 cell line, for instance the CHO-K1 cell line referenced at the ATCC under the number CCL-61 is in the form of a suspension of cells. For example, such suspension of cells can be obtained by cultivation of the cell line in a serum-free medium.
- Trypsin or more generally any enzyme having a serine protease activity such as pronase, subtilisin, plasmin, or thermolysin can be produced by genetic recombination. Trypsin may be produced in particular by means of transgenic plants (WO 00/05384), yeasts or bacteria (WO 01/55429). For instance, a recombinant trypsin provided by Gibco under the trade name TrypLE Select or by Invitrogen under the trade name TrypLE Express is suitable for the purpose of the invention.
- the media used in the context of the invention including culture medium and infection medium are free of serum of animal origin, preferably are free of any protein of animal origin and still more preferably are free of any component of animal origin.
- the set of expression vectors may also comprises:
- Vectors capable of expressing influenza PB1 , PB2, PA and NP proteins may then be for instance the pVAX1 plasmid (Life technology, Cergy Pontoise, FR).
- the viral suspension may be fragmented using detergents or lipid solvents according to methods well known to those skilled in the art, in order to manufacture, for example, vaccines based on fragmented or split viruses, virosomes, or subunit vaccines containing the flu virus hemagglutinin.
- vaccines based on fragmented or split viruses, virosomes, or subunit vaccines containing the flu virus hemagglutinin are considered to be flu virus- derived products.
- the universal pSP-flu plasmid so developed presents several improvements for easy and rapid influenza genome cloning.
- the recombinant cassette can be used to clone every influenza RNA fragments from type A and B virus.
- Universal pSP-flu plasmid contains three enzymatic sites (Bbsl, Sbfl and Notl) that can be used to remove any residual empty plasmids after the cloning step. Linearization with Bbsl enzyme, containing a cleavage point outside of the recognition site, generated cohesive ends and enabled the recircularization of plasmid.
- PB2 contains the PB2, PA, NP, NS, and M genes from the A PR/8/34 (H1 N1 ) virus, the PB1 gene from the A/California/07/09 (H1 N1 ) virus, the HA chimeric gene A California/07/09-A/PR/8/34 (H1 N1 ) and the NA chimeric gene A California/07/09-A/PR/8/34 (H1 N1 ).
- the A/NC/20/99 (H1 N1 ) virus was used as positive control for each reverse genetics experiment.
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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP13742451.1A EP2880154B1 (en) | 2012-08-03 | 2013-07-29 | Production of infectious influenza viruses |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP12305968 | 2012-08-03 | ||
EP13742451.1A EP2880154B1 (en) | 2012-08-03 | 2013-07-29 | Production of infectious influenza viruses |
PCT/EP2013/065920 WO2014019990A1 (en) | 2012-08-03 | 2013-07-29 | Production of infectious influenza viruses |
Publications (2)
Publication Number | Publication Date |
---|---|
EP2880154A1 true EP2880154A1 (en) | 2015-06-10 |
EP2880154B1 EP2880154B1 (en) | 2019-05-15 |
Family
ID=48900986
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP13742451.1A Active EP2880154B1 (en) | 2012-08-03 | 2013-07-29 | Production of infectious influenza viruses |
Country Status (11)
Country | Link |
---|---|
US (1) | US9982240B2 (en) |
EP (1) | EP2880154B1 (en) |
JP (1) | JP6335167B2 (en) |
KR (1) | KR20150038374A (en) |
CN (1) | CN104704112A (en) |
AU (1) | AU2013298632B2 (en) |
BR (1) | BR112015002268A2 (en) |
CA (1) | CA2880409A1 (en) |
MX (1) | MX360120B (en) |
SG (1) | SG11201500508XA (en) |
WO (1) | WO2014019990A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20160014657A (en) * | 2013-06-06 | 2016-02-11 | 노파르티스 아게 | Influenza virus reassortment |
CN104232594B (en) * | 2014-09-11 | 2017-05-31 | 中国农业科学院哈尔滨兽医研究所 | Recombination classes fowl type H1N1 inactivated influenza virus vaccines strain (JS40/PR8) and its preparation method and application |
CN105754997B (en) * | 2016-03-30 | 2019-02-26 | 华中农业大学 | DRV-AH08 plants of rabies viruses of guide RNA and its application in the drug of preparation prevention and treatment rabies viruses |
Family Cites Families (15)
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DE69638058D1 (en) | 1995-06-15 | 2009-11-26 | Crucell Holland Bv | Packaging systems for human recombinant adenoviruses for gene therapy |
TW389999B (en) | 1995-11-21 | 2000-05-11 | Toshiba Corp | Substrate having shallow trench isolation and method of manufacturing the same |
US6544785B1 (en) | 1998-09-14 | 2003-04-08 | Mount Sinai School Of Medicine Of New York University | Helper-free rescue of recombinant negative strand RNA viruses |
US8715940B2 (en) | 1999-04-06 | 2014-05-06 | Wisconsin Alumni Research Foundation | Method of making recombinant influenza virus |
GB9916794D0 (en) * | 1999-07-16 | 1999-09-22 | Isis Innovation | In vitro virus reconstitution |
GB9923176D0 (en) | 1999-09-30 | 1999-12-01 | Smithkline Beecham Biolog | Novel composition |
KR100848719B1 (en) | 2000-04-28 | 2008-07-25 | 세인트 쥬드 칠드런즈 리써치 호스피탈 | Dna transfection system for the generation of infectious influenza virus |
US7332332B2 (en) | 2001-04-23 | 2008-02-19 | Amaxa Ag | Buffer solution for electroporation and a method comprising the use of the same |
WO2005062820A2 (en) | 2003-12-23 | 2005-07-14 | Medimmune Vaccines, Inc | Multi plasmid system for the production of influenza virus |
CA2559371C (en) * | 2004-03-09 | 2014-07-08 | Chiron Corporation | Influenza virus vaccines |
EP1741778B1 (en) | 2005-07-07 | 2007-12-26 | Amaxa AG | Procedure for the treatment of small volumes with electric current |
US20070111309A1 (en) | 2005-10-04 | 2007-05-17 | Daelli Marcelo G | Vero cell line adapted to grow in suspension |
CN100571775C (en) * | 2006-11-09 | 2009-12-23 | 中国农业科学院哈尔滨兽医研究所 | Avian influenza virus marking vaccine and its production and application |
WO2009152181A1 (en) | 2008-06-11 | 2009-12-17 | Flugen, Inc. | Cell-based systems for producing influenza vaccines |
AU2009272857B2 (en) | 2008-07-15 | 2015-06-18 | Crucell Holland B.V. | Scalable process for culturing PER.C6 cells and producing products therefrom |
-
2013
- 2013-07-29 CA CA2880409A patent/CA2880409A1/en not_active Abandoned
- 2013-07-29 AU AU2013298632A patent/AU2013298632B2/en not_active Ceased
- 2013-07-29 KR KR1020157004966A patent/KR20150038374A/en not_active Application Discontinuation
- 2013-07-29 US US14/419,235 patent/US9982240B2/en active Active
- 2013-07-29 MX MX2015001499A patent/MX360120B/en active IP Right Grant
- 2013-07-29 EP EP13742451.1A patent/EP2880154B1/en active Active
- 2013-07-29 JP JP2015524755A patent/JP6335167B2/en not_active Expired - Fee Related
- 2013-07-29 SG SG11201500508XA patent/SG11201500508XA/en unknown
- 2013-07-29 CN CN201380051942.6A patent/CN104704112A/en active Pending
- 2013-07-29 BR BR112015002268A patent/BR112015002268A2/en not_active Application Discontinuation
- 2013-07-29 WO PCT/EP2013/065920 patent/WO2014019990A1/en active Application Filing
Non-Patent Citations (1)
Title |
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See references of WO2014019990A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP2880154B1 (en) | 2019-05-15 |
US9982240B2 (en) | 2018-05-29 |
MX2015001499A (en) | 2015-08-20 |
AU2013298632A1 (en) | 2015-02-26 |
US20150191703A1 (en) | 2015-07-09 |
CA2880409A1 (en) | 2014-02-06 |
BR112015002268A2 (en) | 2017-11-07 |
AU2013298632B2 (en) | 2018-11-01 |
MX360120B (en) | 2018-10-23 |
JP2015530875A (en) | 2015-10-29 |
SG11201500508XA (en) | 2015-02-27 |
JP6335167B2 (en) | 2018-05-30 |
KR20150038374A (en) | 2015-04-08 |
WO2014019990A1 (en) | 2014-02-06 |
CN104704112A (en) | 2015-06-10 |
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